A method for simultaneous targeted mutagenesis of all nuclear rDNA repeats in Saccharomyces cerevisiae using CRISPR-Cas9

Abstract

Saccharomyces cerevisiae has been the prime model to study the assembly and functionality of eukaryotic ribosomes. Within that vast landscape, the specific problem of mutagenizing all 150 nuclear rRNA genes was bypassed using strains whose chromosomal copies had been deleted and substituted by plasmid-borne rDNA. Work with these strains has produced important insights, but nucleolar structure is altered and such yeast-specific approaches are elaborate and not transferable to most other eukaryotes. We describe here a simple CRISPR-Cas9 based method to place targeted mutations in all 150 chromosomal rDNA repeats in yeast. The procedure per se is not expected to alter the nucleolus and is potentially applicable also to other eukaryotes. Yeast was transformed with a plasmid bearing the genes for Cas9 and for the guide RNA, engineered to target a site in the SSU region. Our mutagenesis plan included insertion of a spliceosomal intron in the normally intronless yeast nuclear rDNA. Despite the potential lethality of cutting all 150 rDNA repeats at the same time, yeast survived the Cas9 attack through inactivation of the cut sites either by point mutations or by inserting the intron, which was spliced out correctly from the rRNA transcript. In each mutant strain the same mutation was present in all rDNA repeats and was stably inherited even after removal of the Cas9 plasmid.

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae

“…Based on our observations, using a variety of templates to repair Cas9 breaks in the rDNA, we suggest that the pathway of CRISPR/Cas9 editing is very similar to one proposed by Muscarella and Vogt (1993) for how cells survive cleavage of the rDNA locus by an endogenously expressed I- Ppo 1 restriction enzyme. This similarity was also recognized by Chiou and Armaleo (2018). I- Ppo I cuts once in each rDNA repeat and nowhere else in the S. cerevisiae genome (Lowery et al 1992); however, surviving clones with mutations or insertions at the cleavage site are readily obtained.…”

“…Here, we edited the yeast rDNA locus directly without the introduction of a selectable marker by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Doudna and Charpentier 2014). Our method to mutagenize rDNA was developed independently but is similar to one previously reported (Chiou and Armaleo 2018) which was intended for a different biological purpose. We altered the origin of replication that is found in each repeat by deleting it entirely, or by replacing the 11-bp A+T-rich origin consensus sequence with a G+C block or other functional origins.…”

Modeling in yeast how rDNA introns slow growth and increase desiccation tolerance in lichens

“…The splicing signals overlap with those of yeast mRNA introns ( Figure 5 ) and thus are expected to be recognized by yeast spliceosomes. CRISPR-Cas9 technology was seen as the only available way to simultaneously insert the intron into 150 repeats of yeast rDNA ( Chiou and Armaleo 2018 ; Sanchez et al 2019 ) because of the powerful selection of CRISPR against unmodified rDNA. Our method was developed to insert introns into the 18S and 25S rRNA genes by Homology Directed Repair (HDR), but we list here for the record also the base-pair substitutions produced through NonHomologous End Joining (NHEJ) during this work (Supplementary File S2).…”

“…The successful use of CRISPR to mutagenize the entire array of rDNA repeats in yeast has been described and discussed ( Chiou and Armaleo 2018 ; Sanchez et al 2019 ). The interesting, albeit peripheral issue of snoRNA interference with Cas9 described in Results is not further discussed here.…”

“…A Northern analysis of intron splicing in the cultured lichen mycobiont C. grayi supports that hypothesis. To test in a model system the effects of nonself-splicing introns on growth and desiccation tolerance, we used CRISPR to transfer a spliceosomal rDNA intron (first on left in Figure 1 ) from the lichen C. grayi into all 150 rDNA repeats of S. cerevisiae ( Chiou and Armaleo 2018 ), which lacks rDNA introns. We inserted the intron at two yeast rDNA sites occupied by introns in C. grayi ( Figure 3 shows the intron inserted at the 18S site).…”